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R L Hill

Publications and source records attributed to R L Hill.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase specific for glycosylation of threonine residues.

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase from porcine submaxillary glands was purified to electrophoretic homogeneity. IgG prepared from antisera against the pure enzyme immunoprecipitated the transferase in Triton X-100 extracts of submaxillary glands. The submaxillary transferase is a membrane-bound enzyme in contrast to the pure bovine colostrum enzyme, which is soluble in the absence of detergents. Both transferases have similar properties but also differ significantly. Examination of the acceptor substrate specificity of the submaxillary gland transferase showed that it specifically transferred N-acetylgalactosamine from UDP-GalNAc to the hydroxyl group of threonine and was devoid of transferase activity toward serine-containing peptides. These results imply that more than one transferase is involved in forming the GalNAc-threonine and the GalNAc-serine linkages found in O-linked oligosaccharides in glycoproteins. The amino acid sequence adjacent to glycosylated threonine residues may influence the rate of glycosylation by the pure transferase. For example, the second threonine residue in the sequence, Thr-Thr, appears to be glycosylated about twice as fast as the first and more rapidly than single, isolated threonine residues. However, no unique consensus sequence for glycosylation of threonine residues is evident, and any accessible threonine residue appears to be a potential acceptor substrate.

Amino Acid Sequence↗
PubMed1992

The binding specificity of normal and variant rat Kupffer cell (lectin) receptors expressed in COS cells.

A receptor uniquely found on the surface of rat Kupffer cells was shown previously to bind oligosaccharides terminating in galactose, N-acetylgalactosamine, and fucose. To analyze further the binding specificity of the receptor, receptor-mediated adhesion of transfected COS cells to immobilized glycolipids of known structure was measured. The glycolipid Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) was the best ligand. Gb5Cer (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) and LacCer (Gal beta 1-4Glc beta 1Cer) bound more weakly (five times less than Gb4Cer) and Gb3Cer (Gal alpha 1-4Gal beta 1-4Glc beta 1Cer), and g3Cer(GalNAc beta 1-4Gal beta 1-4Glc beta 1Cer) bound even more weakly (60 times less than Gb4Cer). Gangliosides did not support adhesion of transfected cells. The adhesion of COS cells transfected with plasmids encoding variants of the receptor was also examined. In each variant, either tryptophan 498 or 523, which are conserved in most C-type lectins, was replaced by one of several amino acids. Variants that retained binding activity had the same specificity as the normal receptor. Differences between variants were noted, however, in maximal levels of adhesion and these differences correlated with altered expression of the receptor variants in COS cells.

Acetylgalactosamine↗
PubMed1992

Distribution and dosimetry of Thorotrast in USUR case 1001.

The distribution of radioactivity and the associated doses were evaluated postmortem for USUR Case 1001, a female who had been injected with Thorotrast some 36 y prior to death. The distribution was determined for four nuclides: 232Th and its decay products, 228Ra and 228Th; and 230Th, a contaminant associated with Thorotrast. More than 90% of the activity was associated with the reticuloendothelial system. Approximately 32% of the total activity was found in the total skeleton (mineral bone and bone marrow), which is somewhat higher than expected from the literature. The 44% found in the liver and 12% in the spleen were somewhat lower than expected. This difference may be attributable, in part, to the initial deposition as influenced by colloidal particle size and to the radiation-induced hyposplenism, splenic atrophy, and slight hepatic atrophy observed at autopsy. In addition, roughly 3% of the activity was found in the Thorotrastoma and surrounding carotid artery tissue. Estimated lifetime absorbed doses from the 232Th series were 15 Gy to the liver, 121 Gy to the spleen, 4 Gy to the skeleton, and 16 Gy to the Thorotrastoma. Comparable dose equivalents to these tissues are 300, 2420, 80, and 320 Sv, respectively, assuming a quality factor of 20 for alpha irradiation.

Aged↗
PubMed1991

Porcine submaxillary mucin contains a cystine-rich, carboxyl-terminal domain in addition to a highly repetitive, glycosylated domain.

The sequence of a 3.65-kilobase cDNA encoding a large portion of the polypeptide chain of porcine submaxillary mucin (apomucin) has been completed. The encoded polypeptide contains 1150 residues with the carboxyl-terminal 240 residues forming a globular domain that is rich in half-cystine, but deficient in sites for oligosaccharide attachment. The remaining 910 residues preceding the half-cystine-rich domain appear devoid of secondary structures, but they are rich in serine and threonine to which the O-linked oligosaccharides are bound. The first 391 residues of apomucin contain several tandemly repeated, identical sequences of 81 residues. Blots of genomic DNA partially digested with restriction nucleases show that at least 25 of these identical repeats are present in apomucin. The amino acid composition of apomucin isolated in the absence of protease inhibitors was shown earlier (Eckhardt, A. E., Timpte, C. S., Abernethy, J. L., Toumadje, A., Johnson, W. C., Jr., and Hill, R. L. (1987) J. Biol. Chem. 282, 11339-11344) to be devoid of half-cystine. In contrast, the amino acid composition of mucin purified in the presence of protease inhibitors contains half-cystine in amounts predicted by the cDNA sequence and also suggests that this mucin has about 25 tandem repeats. Thus, apomucin contains at least 2800 amino acid residues. Moreover, immunoblots of apomucin prepared in the presence or the absence of protease inhibitors, with antibodies specific for the half-cystine-rich domains or the tandem repeat sequences, show that the half-cystine-rich domain is absent in apomucin unless protease inhibitors are present throughout. Both types of mucin, however, contain the highly repetitive sequences. The molecular weight of undegraded apomucin has not been established exactly, but gel filtration in 6 M guanidine hydrochloride suggests that it is considerably higher than 250,000. RNA blot analysis shows that apomucin mRNA is large and polydisperse in accord with the message size necessary to synthesize the large apomucin polypeptide. These structural features of apomucin suggest a model for the structure of the mucin molecule that correlates well with its reported properties.

Amino Acid Sequence↗
PubMed1991

Structure of the gene for a carbohydrate-binding receptor unique to rat kupffer cells.

The gene encoding the rat Kupffer cell receptor for fucose and galactose (Lehrman, M. A., and Hill, R. L. (1986) J. Biol. Chem. 261, 7419-7425 and Lehrman, M. A., Haltiwanger, R. S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7426-7432) has been isolated and characterized. The nucleotide sequence of the 9.7-kilobase gene and of 1 kilobase of 5'-flanking sequence was determined. The 5' end of the receptor coding sequence was determined by primer extension and S1 mapping of the receptor transcript. Knowledge of the transcription start site and the 5'-flanking sequence allowed potential promoter elements to be identified. The Kupffer cell receptor coding sequence is encoded by seven exons that are split by six introns. The divisions among domains in the receptor can be correlated with divisions among exons in the gene. The carbohydrate recognition domain is encoded by three exons similar to those in the rat hepatic lectin, human Fc receptor for IgE, and rat cartilage proteoglycan core protein genes. A model for the evolution of the vertebrate lectin genes that have been analyzed is presented.

Amino Acid Sequence↗
PubMed1991

Minimal dose requirements for nasal mupirocin and its role in the control of epidemic MRSA.

Staphylococci are still a leading cause of hospital infection. The success of nasal mupirocin for the control of epidemic methicillin-resistant Staphylococcus aureus (EMRSA), the prevention of colonization of central venous cannulae, and the prevention of septicaemia in haemodialysis patients should encourage the use of minimal dose regimens to minimize the emergence of mupirocin resistance. Mupirocin applied to the anterior nares 4-times daily usually eliminates S. aureus, including EMRSA, within 48 h. Elimination is sustained for several weeks in patients and staff. We recently found that a single dose, or a regimen of 4-times daily for 2 days, eliminated nasal carriage of S. aureus within 24 h; 7 days after a single dose, 92% of the subjects were still cleared; 7 days after the 2-day course, 96% remained free of nasal S. aureus. Ward personnel who are nasal carriers of EMRSA can, provided that other carriage sites are negative, return to work after 2 days of a 4-times daily intranasal regimen. The UK guidelines, recently published in this Journal, recommend an aggressive approach to identifying and eliminating EMRSA, including the elimination of nasal carriage. This approach is increasingly associated with the control of EMRSA in the UK and elsewhere.

Administration, Intranasal↗
PubMed1991

Reduction in the colonization of central venous cannulae by mupirocin.

In an in-vitro simulation of an intravascular cannula enclosed in a fibrin sheath, 0.03 mg1(-1) of mupirocin prevented significant colonization [greater than 15 colony forming units (cfu)] by two clinical isolates of Staphylococcus epidermidis and one each of S. saprophyticus, S. hominis and S. haemolyticus. This suggests that in vivo, where protein binding of mupirocin is 95-97%, 1 mg 1(-1) of mupirocin at the cannula surface would be required to prevent colonization. These results support the findings of our previously published prospective controlled trial, in which mupirocin applied to the insertion sites of 172 internal jugular cannulae reduced the rate of colonization of cannula tips to 5%, compared with 25% for the 186 controls (P less than 0.001). Of the 46 colonized cannula tips from 110 control patients, the same species was isolated from the skin of the insertion site in 67% and from the lumen flush in only 15%. Analysed by patient, mupirocin reduced the proportion of patients with colonized tips from 17% to 3% after 24 h of infection, and from 35 to 10% after 48 h (P = 0.002). The use of agar containing charcoal, as a mupirocin neutralizer, and the incubation of tip-culture plates flooded with the Oxford staphylococcus, gave no evidence of carry over of mupirocin onto cannulae removed from mupirocin-treated patients.

Administration, Topical↗
PubMed1990

Sequences of the CyIIIa actin gene regulatory domain bound specifically by sea urchin embryo nuclear proteins.

Expression of the CyIIIa cytoskeletal actin gene is a marker of differential gene activation in the aboral ectoderm of the early sea urchin embryo. Gene transfer experiments have defined a 2,300 nucleotide cis-regulatory domain required for the correct spatial and temporal control of this gene. This domain includes at least 20 sites at which relatively stable DNA--protein complexes form in vitro on reaction with embryo nuclear extracts. We report the nucleotide sequence of the whole regulatory domain and map the sites at which high-specificity DNA--protein interactions occur. These were located initially by gel shift assays carried out on progressive restriction digests of given subfragments of the large regulatory domain and were located more exactly by oligonucleotide gel shift competitions. Eight of the sites of specific interaction are unique within the CyIIIa regulatory domain, and the remainder consist of five different sites that occur more than once. We observe some well known sequences also found in regulatory regions of other genes, e.g., "CCAAT" and "octamer" elements. The various sites have been classified regarding putative biological function in other work, and the present studies permit an assessment of the number and complexity of interactions constituting each functional class and of the relative locations of sites of each class.

Actins↗
PubMed1990

Mupirocin for the reduction of colonization of internal jugular cannulae--a randomized controlled trial.

In a prospective study, 218 cardiothoracic patients, in whom 'Abbocath-T' cannulae had been inserted preoperatively into the internal jugular vein, were randomized to receive skin preparation of the insertion site with tincture of iodine (108 controls) or tincture of iodine followed by application of sterile 2% calcium mupirocin ointment (110 test patients). Cannulae were usually removed within 48 h of the operation. Patients receiving mupirocin were less likely to develop significant colonization of one or more of their cannulae as judged by Maki's criterion of a yield of greater than 15 colony forming units (cfu) from a cannula segment rolled on an agar plate (17% of mupirocin treated patients compared with 54% of the controls, P less than 0.001). Coagulase-negative staphylococci, micrococci, or both, were the commonest isolates and were cultured from 70% of the 186 control cannulae compared with 24% of 172 cannulae inserted through mupirocin-treated skin (P less than 0.001). A count of more than 15 cfu was found on the tips of 25% control cannulae compared with 5% of the cannulae from mupirocin-treated patients, an effect which was independent of in-situ time (P less than 0.001). For cannulae with colonized tips, the same species was isolated from the skin of the insertion site in 67%, from the exterior of the hub in 61% and from the lumen in only 15%. There were no side effects attributed to mupirocin or superinfection with resistant organisms. We conclude that in cardiothoracic patients the application of mupirocin after standard skin preparation with tincture of iodine significantly reduces the colonization of central venous cannulae by organisms derived from the skin insertion site.

Adolescent↗
PubMed1990

Human cutaneous response to a mixed surfactant system: role of solution phenomena in controlling surfactant irritation.

Exposure of the skin to surfactant-based products can result in irritation. To control this effect researchers are probing mechanisms of surfactant action. In vitro studies show that mixing surfactants often results in less denaturation (swelling) of stratum corneum. We have explored the in vivo human irritation response (using a 21-day cumulative irritation test) to two of these surfactants-sodium lauryl sulfate (SLS) and (C12-C14) alkyl, 7-ethoxy sulfate (AEOS-7EO). Results demonstrate that addition of AEOS-7EO to a constant dose of SLS results in a significant reduction in erythema, hence producing a milder system. The reason for the synergism is unclear, but may related to experimentally determined alterations in the micellar solution properties of the SLS upon addition of AEOS-7EO.

Administration, Cutaneous↗
PubMed1990

Nasal carriage of MRSA: the role of mupirocin and outlook for resistance.

Since the first attempts (1) to eradicate nasal carriage of Staphylococcus aureus with local applications of penicillin, many other topical and systemic antimicrobial agents have been tested, all with limited success. More recently, mupirocin has been much more successful and, in a controlled trial, nasal carriage of S. aureus was eliminated in all subjects and when re-colonisation eventually took place, only 29% had relapsed with their pre-treatment strain. During an MRSA outbreak at a London hospital, standard infection control measures failed to prevent colonisation and infection of more than two hundred patients, but the use of mupirocin was associated with epidemiological control. Of forty patients and thirty-two staff studied, 98.6% of staff and 90.1% of patient-weeks were free of nasal MRSA after treatment. Although resistance to at least 40 mg/l of mupirocin can be produced in vitro and resistant S. aureus have been isolated from patients undergoing prolonged skin treatment with mupirocin, there has been no evidence for the emergence of mupirocin resistance as a mechanism for the relapse of nasal carriage.

Anti-Bacterial Agents↗
PubMed1990

The effect of mouse euthanasia technique on subsequent lymphocyte proliferation and cell mediated lympholysis assays.

The purpose of this study was to determine the effects that specific euthanasia methods have on mitogen induced lymphocyte proliferation (LP) and the induction of alloantigen specific cytolytic T-lymphocytes (CTL). Mice were euthanatized by cervical dislocation (CD), or anesthesia with methoxyflurane or pentobarbital followed by CD (M-CD or P-CD respectively), CO2 overexposure (CO2-OD) or halothane overexposure (H-OD). Mitogenic lymphoproliferation was increased in cells derived from mice euthanatized by M-CD and P-CD. In contrast, the cytolytic profile of CTL derived from mice euthanatized by P-CD, CO2-OD and H-OD was decreased. The results of this study show that euthanasia techniques involving the use of methoxyflurane, pentobarbital, CO2 and halothane affect in vitro lymphoproliferation and CTL function. We conclude that the method of euthanasia influences certain immunologic parameters and selection of a particular technique should be given careful consideration.

Animals↗
PubMed1988

Molecular cloning and sequencing of a cDNA for a carbohydrate binding receptor unique to rat Kupffer cells.

cDNA comprising the entire length of the rat Kupffer cell receptor (Mr = 88,000 and 77,000) for carbohydrates with an affinity for fucose and galactose was isolated, and its nucleotide sequence was determined. Receptor cDNA encoded a protein containing 550 amino acid residues with a Mr = 61,104. This Mr was consistent with the size of the deglycosylated receptor which was found to contain two polypeptides by gel electrophoresis with Mr = 58,000 and 52,000, respectively. Edman degradation of the receptor yielded a sequence which corresponded to amino acid residues 83-104 of the sequence derived from the cDNA. This confirmed that the cDNA which had been isolated corresponded to mRNA for the receptor and suggested that the smaller polypeptide in receptor preparations arises by proteolysis of the intact receptor. Amino acid composition of the receptor was nearly identical to that predicted by the cDNA. The Kupffer cell receptor was found to be homologous to other carbohydrate binding proteins including the hepatic receptors with different binding specificities. The Kupffer cell receptor also contained a series of 18 contiguous, homologous sequences with an average length of 14 residues.

Amino Acid Sequence↗
PubMed1988

The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands.

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.

Acetylgalactosamine↗
PubMed1988

Porcine submaxillary gland apomucin contains tandemly repeated, identical sequences of 81 residues.

A lambda gt11 cDNA library, prepared from porcine submaxillary gland mRNA, was screened with anti-apomucin IgG, and five antibody-reactive phage were isolated. The phage with the largest cDNA insert, designated lambda PSM103, was further characterized. Its fusion protein reacted with anti-apomucin IgG and was used to affinity purify antibodies that specifically reacted with apomucin, indicating that the protein shares antigenic determinants with apomucin. The nucleotide sequence of 1510 bases in the 3.7-kilobase cDNA insert of lambda PSM103 has been established, thereby giving a deduced amino acid sequence of 503 residues in apomucin, or about 45% of the molecule. The deduced sequence of the apomucin polypeptide was found to contain 4.8 tandemly repeated, identical sequences of 81 residues each. The presence of these uniquely repeated sequences was confirmed by restriction endonuclease digestion of DNA derived from lambda PSM103. The repeat sequence was also confirmed in apomucin by the isolation of an 81-residue tryptic peptide with an amino acid composition and an amino-terminal amino acid sequence (up to 44 residues) identical to those of the tandem repeat. Moreover, the peptide was isolated in 760% yield, indicating that the tandem repeat occurs at least eight times in apomucin. The presence of such a long repetitive region in the gene for apomucin raises the possibility for considerable polymorphism in the gene and a corresponding size heterogeneity of apomucin. The predicted secondary structure of the 503 residues confirms the earlier proposal that apomucin is an extended, nonglobular polypeptide. Although the sequences around 192 serine and threonine residues have been established in apomucin, a recognition sequence for the N-acetylgalactosaminyltransferase that initiates glycosylation of apomucin is not evident, except that the glycosylated residues occur in turns.

Amino Acid Sequence↗
PubMed1988

Elimination of nasal carriage of methicillin-resistant Staphylococcus aureus with mupirocin during a hospital outbreak.

During a hospital outbreak of methicillin-resistant Staphylococcus aureus (MRSA), involving more than 200 patients, 40 patients and 32 hospital staff who were stable nasal carriers of MRSA received topical application of 2% mupirocin, formulated in a white soft paraffin and lanolin ointment, to their anterior nares for five days. Nasal carriage was eliminated in all patients and staff, usually within the first 48 h of treatment. Of the 40 patients, 36 remained clear of nasal MRSA for the duration of their follow-up (mean = 2 weeks) and four became re-colonized one to five weeks after their course. Immediately after the course, the number of patients with MRSA isolated from wounds and wrists fell from 16 to 7, and from 16 to 3, respectively. Of the 32 staff, all were negative one week after the course, and of the 22 still available for follow-up at eight weeks, all were consistently negative (mean period of follow-up = 7.8, range = 1-20 weeks). Four patients and five staff were re-colonized with MRSA between one to five, and two to twelve weeks, respectively, after treatment. Overall, in the post-treatment follow-up, 98.6% of the staff-weeks and 90.1% of the patient-weeks were free of nasal MRSA. MICs of mupirocin for both pre and post treatment isolates were all 0.03 or 0.06 mg/l. The elimination of nasal MRSA by mupirocin, and the introduction of isolation facilities, were associated with the control of the outbreak.

Adult↗
PubMed1988

Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo.

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (Kr) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of Kr range from approximately 2 x 10(4) to approximately 2 x 10(6) for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10(5) to 2 x 10(6) molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., less than or equal to 10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage.

Animals↗